Yeast Two Hybrid system - (Apr/25/2014 )

Good morning everyone !!

To study the interaction between two bacterial proteins, I´m actually doing (or should I say I´m trying to do ) yeast two hybrid assays. This technique is not used in my lab (at least, not from a long time), so no one can advise me . So, I need your help, to know if some one of you have already had the opportunity to do Y2H assays, so he can may be have the extreme kidness to give me a protocol that works. I´m using the AH109 strain (the most communally used strain in Y2H... I think). My problem is that I can not get blue colonies in minimal medium without tryptophan and leucine, or even white colonies , so I have some doubts about the protocol of the Middle Ages that I´m using. I should say that my plasmid constructions (pGADT7 containig N-terminal fusion of proteinX to GAL4-AD and pGBKT7 containing N-terminal fusion of proteinY to GAL4 DNA-BD) are already prepared, I just need the steps for yeast transformation and media preparation.

 

THANK YOU SO MUCH for your reply !

But what is the protocol that you use?

We can not help you with your protocol if you don't tell us what it is...

Giving a complete different (new) protocol might not solve your problem...

 

microbiologiste24 on Fri Apr 25 09:07:37 2014 said:

Good morning everyone !!

To study the interaction between two bacterial proteins, I´m actually doing (or should I say I´m trying to do ) yeast two hybrid assays. This technique is not used in my lab (at least, not from a long time), so no one can advise me . So, I need your help, to know if some one of you have already had the opportunity to do Y2H assays, so he can may be have the extreme kidness to give me a protocol that works. I´m using the AH109 strain (the most communally used strain in Y2H... I think). My problem is that I can not get blue colonies in minimal medium without tryptophan and leucine, or even white colonies , so I have some doubts about the protocol of the Middle Ages that I´m using. I should say that my plasmid constructions (pGADT7 containig N-terminal fusion of proteinX to GAL4-AD and pGBKT7 containing N-terminal fusion of proteinY to GAL4 DNA-BD) are already prepared, I just need the steps for yeast transformation and media preparation.

 

THANK YOU SO MUCH for your reply !

Thank you, for your answer Pito,

The problem is that I got few white colonies and only in the SD media with tryptophane and leucine, here is my protocol :

 

Stock solution :
SorB: 100 mM lithium acetate + 10 mM Tris-Hcl (pH 8.0) + 1 mM EDTA (pH 8.0) + 1 M sorbitol, adjusted with acetic acid to pH 8,0.
Carrier DNA: 10 mg/ml salmon sperm DNA (Invitrogen) denaturated at 100ºC for 10 min and cooled on ice.
PEG: 100 mM lithium acetate + 10 mM Tris-Hcl (pH 8,0) + 1 mM EDTA (pH 8.0) + 40% PEG3350.

 

Day 1: Pre-culture
1- Grow Saccharomyces cerevisiae AH109 O/N in 25 ml of YPAD, 30ºC, shaking 200 rpm.

 

Day 2: Preparation of competent cells (cells kept at RT)
2- Inoculate 50 ml of YPAD with the O/N culture (intial OD600nm 0,1).
3- Grow at 30ºC, shaking 200 rpm to OD600nm 0,5-0,6 (it should take between 4 and 6h).
4- Centrifuge in 50 ml Falcon tubes (3 min / 2000 rpm or 500 g), discard supernatant.
5- Wash cells with 15 ml sterile H2O, centrifuge (3 min / 2000 rpm or 500 g), discard supernatant.
6- Wash cells with 10 ml SorB, centrifuge (3 min / 2000 rpm or 500 g), discard supernatant.
7- Dissolve cells in 360 µl of SorB and add 40 µl carrier DNA.
8- In nine 1,5 ml µtubes put: 12 µl of these competent cells and 1 µl of each plasmid (see the combinations), mix by flicking.
Plasmids :
pDK20 N-terminal fusion of proteinX to GAL4-AD
pDK21 N-terminal fusion of proteinX to GAL4-BD
pDK22 N-terminal fusion of proteinY to GAL4-AD
pDK26 N-terminal fusion of proteinY to GAL4-BD
→ pGADT7 contain the transcriptional activation domain GAL4-AD (Leucin marker).
→ pGBKT7 contain the DNA binding domain GAL4 DNA-BD (Tryptophan marker).
Combinations:
pDK20 + pDK21 / pDK22 + pDK26 / pDK20 + pDK26 / pDK21 + pDK22/ pDK20 alone/ pDK21 alone / pDK22 alone / pDK26 alone / without plasmids.
9- Add 6-fold volume of sterile PEG, mix by flicking.
10- Incubate 30 min at 30ºC.
11- Heat shock 15 min at 42ºC (waterbath).
12- Centrifuge 3 min / 2000 rpm or 500 g, suck off supernant.
13- Wash cells once with 1 ml YPAD, centrifuge 3 min / 2000 rpm or 500 g, discard the supernant.
14- Resuspend cells in 1 ml YPAD.
15- Incubate in a shaker (200 rpm) for 4-6 h at 30ºC.
16- Spread 2,5µl, and 100µl on plates.
Plate on + X-Gal (80 µg/ml)

 

Selects for Phenotype
SD-medium - White colonies
SD-medium - Trp pGBKT7 White colonies
SD-medium - Leu pGADT7 White colonies
SD-medium - Leu - Trp pGADT7 and pGBKT7 Blue colonies

Its of course hard to state what is wrong, but I, for example, don't wash cells with sterile H20 nor do I wash them with SorB.

 

I just spin the cells down (step 4) for 1 minute at 7500 RPM, pipette of the supernatants as good as possible. Then I add a LiAC/TE mixture (100µl LiAC 1M, 100µl TE 10x and 800 µl miliQ) 1ml per pellet (per 50ml) 

After this I simple take 0,1ml of this 1ml (its more with the pellet and some left over of the medium) and put this in a new tube (containing the DNA I use to transform).

In this tube I then add 10µl carrier DNA (10mg/ml). I incubate this at room temperature for 30 minutes and than add a PEG/LiAC/TE mixture. Followed with the incubation and heatshock.

I also don't wash the cells and so on..

After the heat shock I centrifuge the cells, remove the supernatants and add demiwater and plate it...

 

 

Are you sure your plasmids contain the genes for the blue-white screening? Because the commercial vectors do not contain this (if I am not mistaken).

 

Also this part is a bit weird to me:

 

 

 

microbiologiste24 on Mon Apr 28 06:09:07 2014 said:

Thank you, for your answer Pito,

The problem is that I got few white colonies and only in the SD media with tryptophane and leucine, here is my protocol :

 

Stock solution :
SorB: 100 mM lithium acetate + 10 mM Tris-Hcl (pH 8.0) + 1 mM EDTA (pH 8.0) + 1 M sorbitol, adjusted with acetic acid to pH 8,0.
Carrier DNA: 10 mg/ml salmon sperm DNA (Invitrogen) denaturated at 100ºC for 10 min and cooled on ice.
PEG: 100 mM lithium acetate + 10 mM Tris-Hcl (pH 8,0) + 1 mM EDTA (pH 8.0) + 40% PEG3350.

 

Day 1: Pre-culture
1- Grow Saccharomyces cerevisiae AH109 O/N in 25 ml of YPAD, 30ºC, shaking 200 rpm.

 

Day 2: Preparation of competent cells (cells kept at RT)
2- Inoculate 50 ml of YPAD with the O/N culture (intial OD600nm 0,1).
3- Grow at 30ºC, shaking 200 rpm to OD600nm 0,5-0,6 (it should take between 4 and 6h).
4- Centrifuge in 50 ml Falcon tubes (3 min / 2000 rpm or 500 g), discard supernatant.
5- Wash cells with 15 ml sterile H2O, centrifuge (3 min / 2000 rpm or 500 g), discard supernatant.
6- Wash cells with 10 ml SorB, centrifuge (3 min / 2000 rpm or 500 g), discard supernatant.
7- Dissolve cells in 360 µl of SorB and add 40 µl carrier DNA.
8- In nine 1,5 ml µtubes put: 12 µl of these competent cells and 1 µl of each plasmid (see the combinations), mix by flicking.
Plasmids :
pDK20 N-terminal fusion of proteinX to GAL4-AD
pDK21 N-terminal fusion of proteinX to GAL4-BD
pDK22 N-terminal fusion of proteinY to GAL4-AD
pDK26 N-terminal fusion of proteinY to GAL4-BD
→ pGADT7 contain the transcriptional activation domain GAL4-AD (Leucin marker).
→ pGBKT7 contain the DNA binding domain GAL4 DNA-BD (Tryptophan marker).
Combinations:
pDK20 + pDK21 / pDK22 + pDK26 / pDK20 + pDK26 / pDK21 + pDK22/ pDK20 alone/ pDK21 alone / pDK22 alone / pDK26 alone / without plasmids.
9- Add 6-fold volume of sterile PEG, mix by flicking.
10- Incubate 30 min at 30ºC.
11- Heat shock 15 min at 42ºC (waterbath).
12- Centrifuge 3 min / 2000 rpm or 500 g, suck off supernant.
13- Wash cells once with 1 ml YPAD, centrifuge 3 min / 2000 rpm or 500 g, discard the supernant.
14- Resuspend cells in 1 ml YPAD.
15- Incubate in a shaker (200 rpm) for 4-6 h at 30ºC.
16- Spread 2,5µl, and 100µl on plates.
Plate on + X-Gal (80 µg/ml)

 

Selects for Phenotype
SD-medium - White colonies
SD-medium - Trp pGBKT7 White colonies
SD-medium - Leu pGADT7 White colonies
SD-medium - Leu - Trp pGADT7 and pGBKT7 Blue colonies

 

First of all, Thank you very much for your reply Pito

 

So, for the Blue-white screening: the gene which encode for one of my proteins is cloned into a vector containing the DNA binding sequence (pGBKT7 plasmid), and the other into a vector containing the transcription activation sequence (pGADT7 plasmid) . So after the co-transformation, only if my proteins X and Y physically interact with one another, the DNA Binding Domain and Activation Domain brought together to reconstitute a functionally active factor that binds to upstream specific sequences of the reporter gene (His3, URA3, ADE2, MEL1 and lacZ) and activates their expression. MEL1 encode α-galactosidase. Because α-galactosidase is a secreted enzyme, it can be assayed directly on X-α-Gal indicator plates, which employ blue/white screening. As lacZ encode for β-galactosidase do you think that I can have blue colonies if I add X-β-Gal in the plates ?!

 

Also, the pGADT7 plasmid contain the gene encoding for Leucine, and pGBKT7 plasmid contain the gene encoding for Tryptophane..... oups ! sorry if the yeast cell receive both plasmids I can have blues colonies in all the mediums and not just in SD-medium - Leu - Trp, sorry for this fault.

 

- What do you mean by: 1ml per pellet (per 50ml). Do you use 50 ml of yeast culture for the co-transformation of each plasmid combination ?, because I use only one 50 ml of yeast culture for my nine combinations, may be I am also not using enough cells ?

- Can you tell me what is the TE 10x ?

- Can you also tell me what are the concentrations that you use for your PEG/LiAC/TE mixture ?

 

Thank you in advance

microbiologiste24 on Wed Apr 30 08:09:23 2014 said:

First of all, Thank you very much for your reply Pito

 

So, for the Blue-white screening: the gene which encode for one of my proteins is cloned into a vector containing the DNA binding sequence (pGBKT7 plasmid), and the other into a vector containing the transcription activation sequence (pGADT7 plasmid) . So after the co-transformation, only if my proteins X and Y physically interact with one another, the DNA Binding Domain and Activation Domain brought together to reconstitute a functionally active factor that binds to upstream specific sequences of the reporter gene (His3, URA3, ADE2, MEL1 and lacZ) and activates their expression. MEL1 encode α-galactosidase. Because α-galactosidase is a secreted enzyme, it can be assayed directly on X-α-Gal indicator plates, which employ blue/white screening. As lacZ encode for β-galactosidase do you think that I can have blue colonies if I add X-β-Gal in the plates ?!

 

Also, the pGADT7 plasmid contain the gene encoding for Leucine, and pGBKT7 plasmid contain the gene encoding for Tryptophane..... oups ! sorry if the yeast cell receive both plasmids I can have blues colonies in all the mediums and not just in SD-medium - Leu - Trp, sorry for this fault.

 

- What do you mean by: 1ml per pellet (per 50ml). Do you use 50 ml of yeast culture for the co-transformation of each plasmid combination ?, because I use only one 50 ml of yeast culture for my nine combinations, may be I am also not using enough cells ?

- Can you tell me what is the TE 10x ?

- Can you also tell me what are the concentrations that you use for your PEG/LiAC/TE mixture ?

 

Thank you in advance

 

 

TEx10 is just TE X 10 (so 10 times the working concentration)

 

forthe PEF/LiAC/TE mixture: 200µl TE 10x; 200µl LiAC 1M; 1,6ml 50% PEG

 

For the 1 ml/pellet => I put the 50ml Medium with the cells in an eppendorf 50ml tube (plastic sterile tubes) and centrifuge this , then I remove the supernatans  and add 1 ml LiAC/TE mixture...

 

For the blue white screening: yes, it activates the LacZ if it worked (the interaction). (when using x- gal)

The MEL1 system is pretty much the same , you just add x-alpha gal.

 

 

 

 

I will need to read the rest of your questions later today, I don't have time now.

microbiologiste24 on Wed Apr 30 08:09:23 2014 said:

First of all, Thank you very much for your reply Pito

 

So, for the Blue-white screening: the gene which encode for one of my proteins is cloned into a vector containing the DNA binding sequence (pGBKT7 plasmid), and the other into a vector containing the transcription activation sequence (pGADT7 plasmid) . So after the co-transformation, only if my proteins X and Y physically interact with one another, the DNA Binding Domain and Activation Domain brought together to reconstitute a functionally active factor that binds to upstream specific sequences of the reporter gene (His3, URA3, ADE2, MEL1 and lacZ) and activates their expression. MEL1 encode α-galactosidase. Because α-galactosidase is a secreted enzyme, it can be assayed directly on X-α-Gal indicator plates, which employ blue/white screening. As lacZ encode for β-galactosidase do you think that I can have blue colonies if I add X-β-Gal in the plates ?!

 

Also, the pGADT7 plasmid contain the gene encoding for Leucine, and pGBKT7 plasmid contain the gene encoding for Tryptophane..... oups ! sorry if the yeast cell receive both plasmids I can have blues colonies in all the mediums and not just in SD-medium - Leu - Trp, sorry for this fault.

 

- What do you mean by: 1ml per pellet (per 50ml). Do you use 50 ml of yeast culture for the co-transformation of each plasmid combination ?, because I use only one 50 ml of yeast culture for my nine combinations, may be I am also not using enough cells ?

- Can you tell me what is the TE 10x ?

- Can you also tell me what are the concentrations that you use for your PEG/LiAC/TE mixture ?

 

Thank you in advance

 

 

the gene to check your interaction, this is the MEL1 gene? Or a combination of ...? (whats your reporter gene, not all of them I assume?)

 

The genes for leucine and tryptophane are (I pressume) only used as an indicator that the transformation worked?

 

 

Hi microbiologiste24, I hope that your problem has been solved, I'm doing my PhD in gene regulation, using the DNA -BD Gal4 model that we have in the laboratory, unfortunately the sequence did not correspond with the map, so I'm looking for a vector containing the GAL4 DBD, could you send me the plasmid, I'll pay the postage?

 

thanks